The Wnt Signaling Pathway

Jennifer Slade B.Sc. (Hon)

M.Sc. Candidate

Outline

• Introduction– Overview of Wnt signaling

• Components of Wnt signaling– Wnt proteins

• Palmitoylation

• Transport

– Wnt receptors• Interactions with

extracellular proteins

• How they signal

– Cytoplasmic Signaling cascade

– Nuclear Signaling Cascade– Target genes

• Non-Wnt pathway genes• Feedback loop

• Mutant Wnt Pathway Phenotypes– Wnt Redundancy

• Wnt and Human Disease• Summary

Introduction• Important in multiple developmental events

– Mutated: leads to disease

• Canonical pathway:– Wnt signaling through Frizzled to β-catenin

• Main intracellular proteins involved:– Dishevelled (Dsh) - Glycogen synthase kinase 3 – Axin (GSK-3)– β –catenin - Adenomatous Polyposis Coli

(APC)

• Wnt signaling inhibits degradation of β -catenin• β -catenin interacts with Tf lymphoid enhancer-binding factor

(TCF)

Overview of Pathway

Dsh

Dsh APCGSK3

Axin

Β-c

TCF

Β-c TCF

Axin

Β-c

Β-c

Β-c

WNT protein

Frizzled

LRP5/6

DNA

First Component: Wnt Proteins

Wnt Proteins

• Large family of secreted molecules– 350 to 400 amino acids– Signal sequence– Invariant pattern of 23-24 conserved cysteines

• Name derived from first 2 members discovered:– Drosophila Wingless– Mouse int-1

• Involved in intercellular signaling during development– Early mesodermal patterning of embryo– Morphogenesis of brain and kidneys

Wnt Proteins

• Secreted but insoluble (hydrophobic)– Palmitoylated– Enzyme responsible: porcupine (por) in Drosophila or

mom-1 in C. elegans– Essential for function and signaling

• Mutation of cysteine• Removal of palmitate

• Drosophila homologue – Wingless– Loses hydrophobicity and activity when por eliminated– Por is necessary for lipidation/membrane targeting

Inactive WNTProtein

Palmitoylation

WNT

H-C-CH2-S-H

O=C-O-

HO-C-(CH2)14CH3

O

WNT

H-C-CH2-S

O=C-O-

C-(CH2)14CH3

O

CysteineResidue Palmitic

Acid

PalmitoylatedWNT protein

por mom-1

Function of Palmitoylation• Remains unclear• Experiments in:

– Drosophila• Loss of por • Excess of Wingless • Circumvents the loss of por

– Vertebrates• Excess expression of mutant Wnt• Still some Wnt signaling

• Presence of lipid moiety targets Wnt to the membrane• Absence of lipid is overcome by high concentration of

Wnt protein

Transport of Wnt Proteins

• Secreted from cells• Experiment in Drosophila

– Antibody to Wingless• Significant spread in imaginal discs

• Concentration-dependent long-range morphogenetic signals acting on distant neighbours

• Flies have vessicles in imaginal discs– Argosomes– Might carry Wingless as cargo

Extracellular Binding Partners

• Extracellular enhancer:– HSPG – Heparin-sulfated forms of proteoglycans

• Co-receptor on target cells• Drosophila’s Dally

– Lost or mutated: similar phenotype to wingless mutants

• Extracellular inhibitors:– SFRP – Secreted Frizzled-related protein

• Resembles ligand-binding domain of Frizzled

– WIF – Wnt inhibitory factor• Secreted molecules resembling extracellular portion of receptor

• Might promote signaling through protection of Wnts from degradation

Extracellular Binding of Wnts

Por/mom-1

SFRP

WIF

Second Component: WNT Receptors

Wnt Receptors

• Frizzled (Fz) proteins– Seven transmembrane receptors – Long N-terminal extension

• Cysteine rich domain (CRD)

• Overexpression of Fz: – No Wnt signal– Co-overexpression of Wingless: Signaling– Fz activation is ligand dependent

• Fz forms receptor complex with another single-pass transmembrane protein– LRP (Low density receptor related protein)– Arrow in Drosophila

WNT Receptors

• Derailed– Distinct from Frizzled– Transmembrane tyrosine kinase

• Belongs to RYK subfamily

– Contains WIF domain

• In Drosophila:– Binds Dwnt-5

• Regulator of axon guidance in CNS

– Cytoplasmic kinase domain dispensable

• In Vertebrates:– Wnt4 and Wnt5 implicated in axon guidance

• Wnt4 binds to Fz• Wnt5 receptor remains undetermined

Non-Wnt Proteins that Interact with Wnt receptors

• Dickkopf – Dkk1– Encodes cysteine-rich secreted protein

– Binds Wnt coreceptor LRP6 • If Fz has Wnt bound, can still bind to Dkk1 and LRP5/6 to induce

canonical signaling pathway

– Also binds transmembrane protein Kremen

– Endocytosed, depleting LRP6

• Norrin– Ligand that binds to Fz

– No sequence similarity to Wnt

– Can induce canonical signaling pathway

Wnt Receptors and Non-Wnt Proteins

Dkk

Dkk

N N

CanonicalSignaling

How Wnt Receptors Signal• Frizzled

– Binds to Dishevelled (Dsh)• Ubiquitously expressed

– C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif• Required for Fz signaling

• LRP – Binds to Axin– Cytoplasmic tail has several Pro-Pro-Pro-(SerTrp)Pro motifs

• Phosphorylated upon Wnt binding

• Axin and Dsh: DIX domains– Can heterodimerize– LRP and Fz may promote interaction between Dsh and Axin

Third Component: Cytoplasmic Cascade

Dsh

APCAxin

Axin Β-cGSK3

Canonical Signaling• Absence of Wnt Signaling:

– β-catenin phosphorylated by serine/threonine kinase• Casein Kinase or GSK-3

– Facilitated by scaffolding proteins APC and Axin– Degradation complex

– Recognized by β -TrCP• Ubiquitinates for degradation via proteosome

• Activation of Wnt signaling:– β -catenin levels accumulate– Enter the nucleus to induce transcription of target genes

• Mutant β-catenin (no phosphorylation sites)– Wnt unresponsive – β–catenin active in entering the nucleus– Constitutive Wnt signaling common in neoplasms

Presence of Wnt Signaling

• Three ways of β-catenin accumulation– Disruption of degradation complex

1. Recruitment of Axin to LRP or Fz/Dsh– Amount of Axin in cell much lower than other complex proteins

• Limiting factor

2. Protein phosphatases– PP2A

• Binds to Axin, dephosphorylates GSK-3

3. GBP/Frat– GSK-3 binding protein– Removes GSK-3 from degradation complex

Regulation of Cytoplasmic Cascade

Dsh

APCAxin

Axin Β-cGSK3

PP2A

GBP/Frat

Β-cΒ-c

Β-c

Β-c

Fourth Component: Signaling in the Nucleus

Β-c TCF

DNA

Β-c

Signaling in the Nucleus

• In presence of Wnt binding only– β-catenin enters nucleus– Binds to TCF DNA-binding proteins

• No Wnt: TCF represses gene transcription– Forms complex with Groucho

• Interacts with histone deacetylases

• β-catenin converts TCF to activator– Displaces Groucho– Recruits histone acetylase (CBP – cyclic AMP response

element binding protein)• Co-activator

Other controls of nuclear signaling

• Protein partners– Chibby

• Nuclear antagonist• Binds C-terminus of β-catenin

– ICAT• Blocks binding of β-catenin to TCF• Disassociates TCF/ β-catenin-CBP complex

– Mitogen-activated protein kinase(MAPK)-related protein kinase NLK/Nemo

• Phosphorylates TCF, sending it to the cytoplasm– Sequestered by 14-3-3 binding protein

• Regulated itself by MAPK kinase TAK1

Groucho

Regulation of Nuclear Signaling

Β-c

Β-cΒ-c

Β-c TCF

Groucho

Chibby

ICAT

TCF NLK

TCF14-3-3

P

Fifth Component: Target Genes

Β-c TCF

DNA

Β-c

Target Genes: Non-Wnt Pathway• Many transcription factors and signaling proteins

– Including:

• Members of the homeobox family– Engrailed (en)– Ultrabithorax (Ubx)

• Genes expressed in development of the embryo– Siamois (organizing center)– Achaete (ac – proneural gene)

• Differential control dependent on cellular context• Ac activated in wing imaginal disc, but repressed in eye imaginal disc

• Cellular proliferation genes– Cell cycle regulators

Target Genes: Wnt Pathway Components• Feedback control• Receptor components:

– Frizzled family of receptors• DFz2 in Drosophila down-regulated by wingless

– Number of LRP receptors controlled by Wg signaling

• Cytoplasmic negative regulators– Naked cuticle (naked)

• Encodes protein that binds to Dsh and inhibits Wnt signaling

– Axin2 gene

Β-cTCF

Β-cTCF

Dsh Dshnakednaked

naked

Target Gene Interacts With Effect on Target Gene Expression

Effect on Wnt Pathway

Fz Wnt Down Inactivate

Dfz2 Wnt Down Inactivate

Dfz3 Wnt Up Activate

Fz7 Wnt Up --

Arrow/LRP Wnt and Axin Down Inactivate

Dally Wnt Down --

Wingful/notum HSPG Up Inactivate

Naked Dsh Up Inactivate

Axin2 β-catenin Up Inactivate

β-TRCP β-catenin Up Inactivate

TCF1 TCF Up Inactivate

LEF1 β-catenin Down Activate

Nemo β-catenin and LEF/TCF

Up Inactivate (Drosophila)

Activate (Zebrafish)

Mutant Wnt Pathway Phenotypes• Study knock-outs

– Gene expression pattern correlates with mutant phenotype • Demonstrates Wnt requirement in developmental process

• Wnt3– Expressed in primitive streak in mouse embryo– Wnt3 mutants – gastrulation defects

• Frizzled4– Cerebellar, auditory and esophageal defects

• TCF1– Defects in limb bud development– Mammory and gut tumours

• Many more

Wnt Redundancy

• Knockout both Wnt1 and Wnt3:– Larger area of CNS disturbed (compared to

single knockouts of either)

• Frizzled mutants do not reveal specific Wnt/Fz pairs– Single Fz activated by many Wnts– Single Wnt may bind many Fzs

Wnt Signaling and Human Disease

Gene Disease

Wnt3 Tetra-amelia

LRP5 Bone density defects

Fzd4 Familial Exudative Vitreoretinopathy (FEVR)

Axin2 Tooth agenesis

Predisposition to Colorectal Cancer

APC Familial adenomatous polyposis (FAP)

Colon Cancer

Extracellular Wnt Protein Target Cell Membrane Protein Intracellular Protein

Wnt3 and Tetra-amelia

• Rare human genetic disorder

• Absence of of all four limbs

• Mutated extracellular

Wnt Protein– Loss of function

Wnt3 mutations

LRP and Bone density

• Target cell membrane protein• Mutation of single amino-acid

– Substitution– LRP insensitive to Dkk-mediated Wnt inhibition– Increased bone density of the jaw and palate

• Mutation causing frameshift – Loss of function LRP– Decreased bone density

• Wnt Signaling mediated by LRP– Important in maintenance of normal bone density

Fz4 and FEVR

• Rare eye disease affecting:– The retina – The vitreous– Progressive genetic disease

• Congenital and bilateral• Target cell membrane proteins• Mutations in both Fz4 and LRP

– Frizzled mutated in seventh transmembrane domain– LRP proteins prematurely terminated– Loss of Fz4/LRP signaling

Axin2 and Tooth Agenesis

• Intracellular protein

• Nonsense mutation in Axin2

• Oligodontia– Condition where multiple permanent teeth are

missing

• Mutation in Axin2 also results in pre-disposition to colon cancer

APC and FAP

• Intracellular protein• Truncations in APC

– Aberrant activation of Wnt pathway– Increased cell proliferation and adenomatous lesions

• Autosomal dominantly inherited disease• Hundreds or thousands of polyps in the colon and rectum• Mutations in APC also found in:

– Sporadic colon cancer– Several types of tumours– Hepatocellular carcinoma

Summary• Wnt signaling includes:

– Wnt proteins• Palmitoylated

– Receptors• Frizzled and LRP, Derailed

– Cytoplasm proteins• Dsh, Degradation complex: Axin, APC and GSK-3• β-catenin

– Nuclear proteins• TCF, Inhibitors: Groucho, Chibby, ICAT and NLK

– Target genes include non-wnt developmental genes or wnt pathway components

• Feedback

• Pathway involved in many human diseases

References

• Logan, C.Y. and Nusse, R. 2004. The Wnt Signaling Pathway in Development and Disease. Annu. Rev. Cell. Dev. Biol. 20: 781-810

• Wodarz, A. and Nusse, R. 1998. Mechanisms of Wnt Signaling in Development. Annu. Rev. Cell Dev. Biol. 14:59–88